PROJECT SUMMARY/ABSTRACT Despite its reputation as a toxic and foul-smelling gas, hydrogen sulfide (H2S) is an important signaling molecule that plays a role in nearly every organ in the body. H2S therapy is a growing area of research, with studies showing efficacy in many animal models of disease, including cardiovascular disease, neurological diseases, and cancer. However, outcomes appear to depend heavily on the duration and rate of H2S delivery. To fully understand the physiological roles of H2S, to measure its effects on different organs and systems, and to achieve its therapeutic potential, novel methods for delivering H2S with control over the timing, location, rate, and duration of delivery are needed. The long-term goal of this project is to treat diseases by delivery of exogenous H2S; however, chemical tools must first be developed that will enable controlled delivery. These tools, which will provide methods to probe H2S physiology in a variety of diseases, include both enzyme- triggered H2S-releasing prodrugs (control over timing and location of delivery) and H2S releasing micelles with tunable release rates (control over rate and duration of delivery). Through the following specific aims these new chemical tools will be prepared and tested in a biologically relevant application of H2S in cancer therapy: 1. Synthesize enzyme-triggered H2S prodrugs with high specificity This aim will focus on synthesis of small molecules that release H2S only in the presence of specific enzymes, including proteases, esterases, and azoreductases, all of which are upregulated in response to specific diseases that may benefit from H2S treatment. 2. Develop biodegradable H2S-releasing polymer micelles with tunable release rates In this aim H2S release rate will be controlled using a polymer micelle platform. Micelles were chosen due to the many factors that can be controlled to tune H2S release kinetics, including size, shape, critical micelle concentration, and unimer exchange rates. 3. Use these tools to answer controversial biological questions regarding the roles of H2S in the inhibition/promotion of cancer cell proliferation The role of H2S in cancer is complex?it can either inhibit or promote cancer cell growth depending on the rate, dose, and duration of release. The prodrugs and micelles will be tested as anti-cancer agents to measure how release rate affects toxicity and selectivity toward cancer cells over normal cells. The H2S delivery methods proposed here will increase our understanding of the signaling roles that endogenous H2S plays in mammalian biology. Also, we expect that these strategies for controlling timing, location, rate, and duration of H2S delivery will inspire new methods for controlled delivery of other signaling gases. In summary, the studies proposed here will elevate the therapeutic potential of H2S, furthering a research program that may lead to H2S therapies with low toxicity, few side effects, and high efficacy.